Oil biosynthesis

ABSTRACT

The invention provides a method of producing, in a plant, oil having an erucic acid content above 66% erucic acid, the method comprising (i) expressing in the plant nucleic acid encoding an elongase and nucleic acid encoding an acyltransferase enzyme; and (ii) extracting oil from the plant. Corresponding plants seeds and oils are also provided.

This application claims priority, under 35 U.S.C. § 371, toInternational Patent application No. PCT/GB02/04642, filed Oct. 11, 2002and published in English as WO 03/033713 on Apr. 24, 2003, which claimspriority to Great Britain Patent Application No. GB 0124574.5, filedOct. 12, 2001.

FIELD OF THE INVENTION

The present invention relates to the production of commercially usefuloil in plants. In particular, the invention provides a method forproducing such oil in plants. Also provided are the plants or partsthereof from which the oil is derived, use of the plant, and the oilitself.

BACKGROUND OF THE INVENTION

Plants have long been a commercially valuable source of oil.Traditionally, plant oils were used for nutritional purposes. Recently,however, attention has focused on plant oils as sources of industrialoils, for example as replacements for, or improvements on, mineral oils.Given that oil seeds of commercially useful crops such as Brassica napuscontain a variety of lipids (Hildish & Williams, “Chemical Compositionof Natural Lipids”, Chapman Hall, London, 1964), it is desirable totailor the lipid composition to better suit our needs, for example usingrecombinant DNA technology (Knauf, TIBtech, February 1987, 40-47).

The production of commercially desirable specific oils in plants on alarge scale is limited in two ways. Some plant species make oils withvery high levels of essentially pure, specific fatty acids, but thesespecies are unable to be grown in sufficient quantities and ofsufficient yield to provide a commercially valuable product. Other plantspecies produce sufficient amounts of oil, but the oil has low levels ofthe specific desired fatty acids. Nevertheless, the field of oilmodification in plants is wide and a number of different products havealready been designed. Rape oil containing lauric acid has beenmarketed, and soybeans with modified levels of unsaturated fatty acidsare available. In some cases the production of speciality oils seems tobe straight-forward. In others, however, a number of unexpectedcomplications have arisen which have hampered the production of plantscapable of making some specific oils. For example, mutations in plantlipid synthesis genes are generally difficult to detect due to thepleiotrophic effects of mutations on plant hardiness and yield. Even ifdetected, proteins involved in pathways of interest have proveddifficult to isolate due to their biochemical instability. Whereregulation of such proteins has been successfully altered, resultsgenerally do not coincide with expectations, presumably due to theeffect of multiple converging pathways. Examples of such problemsrelating to the production of Arabidopsis producing petroselinic acidare disclosed in Ohlrogge, 13^(th) International Symposium on PlantLipids, Seville, Spain: 219 & 801, (1998). Thus, there is considerablework yet to be done in achieving reliable, large-scale production of arange of commercially desirable oils.

Broadly speaking, there are two main approaches to altering the lipidcontent of an oil, which to date have been applied as alternatives.Firstly, plants may be modified to produce fatty acids which are foreignto the native plant. For example, rape may be modified to producelaureate which is not naturally produced by that plant. Secondly, thepattern and/or extent of incorporation of fatty acids into the glycerolbackbone of a lipid may be altered.

Lipids are formed by the addition of the fatty acid moieties into theglycerol backbone by acyltransferase enzymes. There are three positionson the glycerol backbone at which fatty acids may be introduced. Theacyltransferase enzymes which are specific for each position are hencereferred to as 1-, 2-, and 3-acyltransferase enzymes respectively.

One of the aims of lipid engineering is to produce oils which are highin erucic (22: 1) acid. Such oils are desirable for a number of reasons,in particular as replacements and/or substitutes for mineral oils, asdescribed above. In the case of Brassica napus one of the mostcommercially important crops cultivated today, and other oil seedBrassica species, e.g. Brassica juncea, the 2-acyltransferase positivelydiscriminates against the incorporation of erucic acid in the secondposition. Thus, even in those crops where erucic acid is incorporatedinto the first and third positions, only a maximum of 66% of the fattyacids of the lipid can be erucic acid. These latter varieties of rapeare nevertheless known as HEAR (high erucic acid rape) varieties.

It is desirable to further increase the erucic acid content of both HEARvarieties, and other useful vegetable oil crops, for example maize,sunflower, soya, mustards and linseed. Genes encoding 2-acyltransferaseshave been introduced into plants, in order to try to incorporate erucicacid into the second position of the glycerol backbone, with the aim ofincreasing the overall erucic acid content in a lipid (Brough et al.,Mol Breeding 2: 133-142 (1996)). This was successful in there-distribution of erucic acid in the triglyceride but has not increasedthe overall erucic acid content of the oil. One possible reason for thisis that the levels of “free” erucic acid available for incorporationinto lipids in a plant are too low to support high levels of trierucinsynthesis (Millar et al., The Plant J. 12(1) 121-131 (1997)). Thus,knowledge of the factors involved in the regulation of erucic acidlevels in a plant is being sought.

In this text, the terms “free” or “available” erucic acid mean erucicacid which has not been incorporated into lipid. References to theerucic acid content of oil means that which has been incorporated intolipid.

Biochemical and genetic studies have elucidated most of the pathwaysinvolved in the production of vegetable oils (Ohlrogge & Browse, PlantCell 7: 957-970, 1995). An enzyme involved in the synthesis of fattyacids is the fatty acid elongation enzyme (FAE) complex, also referredto as an elongase. This enzyme complex is responsible for the conversionof fatty acids 18 carbons long, such as oleic (18:1) acid, to fattyacids known as very long chain fatty acids, which include erucic acid(22: 1). Given its involvement in the production of erucic acid, it isapparent that the elongase plays a role in regulation of the levels offree erucic acid in a plant. Thus, it has been suggested that, inrelation to Arabidopsis, over-expression of the FAE1 gene may assist inobtaining higher levels of free erucic acid (Millar et al., The Plant J.12(1) 121-131 (1997)). Depending upon the plant species, the products ofthis enzyme are C20 and C22 saturated fatty acids, utilised in waxproduction in leek, or C20 to C24 monounsaturated fatty acids utilisedas seed storage oils in crucifers.

Over recent years, a number of β-keto-acyl-CoA synthetase (“elongase”)genes, in addition to the Arabidopsis FAE1 gene, have been cloned from avariety of species. Sequence data is available for elongases isolatedfrom Arabidopsis (Millar & Kunst, Plant J. 12: 121-131, (1997)), jojoba(Lassner et al, Plant Cell 8: 281-292, (1996)), honesty (Millar & Kunst,Plant J. 12: 121-131, (1997)), leek (Evenson & Post-Beittenmiller, PlantPhysiol. 109: 707-716, (1995)) and oilseed rape (Sequence: GenbankAF009563 & BNU50771). These enzymes all produce a range of other verylong chain fatty acids besides erucic acid, for example fatty acid 20:1in Arabidopsis and oilseed rape, fatty acids 20:0 and 22:0 in leek andfatty acid 24:1 in honesty and jojoba.

Recently, experiments have been performed on high-erucic acid rapeseedplants in which the plants were transformed with constructs encoding anacyltransferase and an elongase. However, none of the transformants werefound to contain erucic acid levels greater than 60% (Han et al., PlantMol. Bio. 46 229-239 (2001)).

SUMMARY OF THE INVENTION

The present invention aims to overcome or ameliorate the above problemsof the prior art by enabling for the first time the production of lipidwith higher levels of erucic acid.

Thus, in a first aspect of the present invention, there is provided amethod of producing, in a plant, oil containing more than 66% erucicacid, wherein the method comprises (i) expressing in the plant nucleicacid encoding an elongase and nucleic acid encoding an acyltransferase;and (ii) extracting oil from the plant.

The present invention is based upon the inventors' discovery that thecombination of an elongase enzyme and an acyltransferase enzymeexpressed in a plant can have the surprising effect of increasing theerucic acid content of oil to above the theoretical maximum of 66%.These results mean that for the first time it has been possible tointroduce erucic acid into all three positions in the glycerol backbonein at least a portion of the lipid of the oil and also cause an overallincrease in the erucic acid incorporated into lipid rather than merelyredistributing the erucic acid on the glycerol backbone. Thus, for thefirst time the invention enables the production of oil containing above66% erucic acid in an ergonomic and agronomic fashion. The resulting oilcan be used in a variety of industrial applications, such as feedstock'sfor surfactants, plasticisers, and surface coatings.

The nucleic acid sequences of the invention may be DNA or RNA, or anyother option. The nucleic acid may be recombinant or isolated.

The elongase enzyme expressed in the plant is preferably one capable ofthe production of very long chain fatty acids including erucic acid,most preferably specific for the production of erucic acid. Examples ofpreferred elongase enzymes are Brassica napus FAE1-1 and FAE1-2 of FIGS.1 and 2 respectively, or elongase enzymes encoded by the B. napus cDNAsequeneces disclosed in WO96/13582, or encoded by the B. napus sequencesGenbank accession nos. AF009563 and BNU50771, or similar enzymes fromother Brassica species, Arabidopsis (Millar & Kunst, Plant J, 12:121-131, (1997), jojoba (Lassner et al, Plant Cell 8: 281-292, (1996)),Lunaria (Millar & Kunst, Plant J. 12: 121-131, (1997)) and Nasturtium(WO95/15387). Also included for use in the present invention are enzymeswhich are substantially identical in sequence to the elongases of FIG. 1and/or FIG. 2, and which share the same enzyme activity as one or moreof the elongases mentioned above.

The acyltransferase enzyme to be expressed in the plant may be a 1-, 2-,or 3-acyltransferase, but most preferably is a 2-acyltransferase, and istherefore capable of introducing a fatty acid into the second positionof the glycerol backbone. More preferably, the 2-acyltransferase iscapable of introducing erucic acid into the glycerol backbone. Examplesof suitable 2-acyltransferase enzymes include those from Limnanthes alba(WO95/27791), Limnanthes douglassi (WO96/24674 and WO96/09394). Alsoincluded are acyltransferases which are substantially identical thereto,and which share the same enzyme activity as one or more of theacyltransferases mentioned above.

BRIEF DESCRIPTION OF THE DRAWINGS

The invention will now be described by way of non-limiting examples,with reference to the drawings in which:

FIG. 1 shows the nucleic acid sequence encoding the Brassica napusenzyme FAE1-1 (SEQ ID NO:1).

FIG. 2 shows the nucleic acid sequence encoding the Brassica napusenzyme FAE1-2 (SEQ ID NO:2).

FIG. 3 shows the nucleic acid sequence encoding the 2-acyltransferase ofLimnanthes douglasii (SEQ ID NO:3).

FIG. 4 shows the construction of plasmid pEW13.

FIG. 5 shows the distribution of erucic acid levels, expressed as mol %of fatty acids, in seeds of LEAR transformants determined by gaschromatography (20:1=eicosenoic acid; 22:1=erucic acid). Transgeniclines designated 333.n and 334.n were transformed with constructscontaining the fae1-1 gene; lines designated 335.n and 336.n weretransformed with constructs containing the fae1-2 gene. Data are derivedfrom single samples of 20 seeds.

FIG. 6 shows the distribution of erucic acid levels, expressed as mol %of fatty acids, in seeds of high erucic acid winter cultivar BGRV2transformants determined by gas chromatography (20:1=eicosenoic acid;22:1=erucic acid). Transgenic lines were transformed with either pEW13(fae1-1 plus lat2) (Lines 337.n, 346.n, 349.n, 360.n, 398.n, 399.n) orpEW14 (fae1-2 plus lat2) (Lines 338.n, 347.n, 350.n, 361.n). Data arederived from single samples of 20 seeds.

DESCRIPTION OF THE INVENTION

In the context of the present invention the term “substantiallyidentical” means that the sequence has at least 50% sequence identity,desirably at least 75% sequence identity and more desirably at least 90or at least 95% sequence identity with one or more of the abovementioned sequences. In some cases the sequence identity may be 99% orabove.

“% identity”, as known in the art, is a measure of the relationshipbetween two polypeptide sequences or two polynucleotide sequences, asdetermined by comparing their sequences. In general, the two sequencesto be compared are aligned to give a maximum correlation between thesequences. The alignment of the two sequences is examined and the numberof positions giving an exact amino acid or nucleotide correspondencebetween the two sequences determined, divided by the total length of thealignment and multiplied by 100 to give a % identity figure. This %identity figure may be determined over the whole length of the sequencesto be compared, which is particularly suitable for sequences of the sameor very similar length and which are highly homologous, or over shorterdefined lengths, which is more suitable for sequences of unequal lengthor which have a lower level of homology.

Methods for comparing the identity of two or more sequences are wellknown in the art. Thus for instance, programs available in the WisconsinSequence Analysis Package, version 9.1 (Devereux J et al, Nucleic AcidsRes. 12:387-395, 1984, available from Genetics Computer Group, Maidson,Wis., USA), for example the programs BESTFIT and GAP, may be used todetermine the % identity between two polynucleotides and the % identitybetween two polypeptide sequences. BESTFIT uses the “local homology”algorithm of Smith and Waterman (Advances in Applied Mathematics,2:482-489, 1981) and finds the best single region of similarity betweentwo sequences. BESTFIT is more suited to comparing two polynucleotide ortwo polypeptide sequences which are dissimilar in length, the programassuming that the shorter sequence represents a portion of the longer.In comparison, GAP aligns two sequences finding a “maximum similarity”according to the algorithm of Neddleman and Wunsch (J. Mol. Biol.48:443-354, 1970). GAP is more suited to comparing sequences which areapproximately the same length and an alignment is expected over theentire length. Preferably, the parameters “Gap Weight” and “LengthWeight” used in each program are 50 and 3 for polynucleotide sequencesand 12 and 4 for polypeptide sequences, respectively. Preferably, %identities and similarities are determined when the two sequences beingcompared are optimally aligned.

Other programs for determining identity and/or similarity betweensequences are also known in the art, for instance the BLAST family ofprograms (Altschul S. F. et al, J. Mol. Biol., 215:403-410, 1990,Altschul S. F. et al, Nucleic Acids Res., 25:289-3402, 1997, availablefrom the National Center for Biotechnology Information (NCB), Bethesda,Md., USA and accessible through the home page of the NCBI atwww.ncbi.nlm.nih.gov) and FASTA (Pearson W. R. and Lipman D. J., Proc.Nat. Acac. Sci., USA, 85:2444-2448, 1988, available as part of theWisconsin Sequence Analysis Package). Preferably, the BLOSUM62 aminoacid substitution matrix (Henikoff S. and Henikoff J. G., Proc. Nat.Acad. Sci., USA, 89:10915-10919, 1992) is used in polypeptide sequencecomparisons including where nucleotide sequences are first translatedinto amino acid sequences before comparison.

Preferably, the program BESTFIT is used to determine the % identity of aquery polynucleotide or a polypeptide sequence with respect to apolynucleotide or a polypeptide sequence of the present invention, thequery and the reference sequence being optimally aligned and theparameters of the program set at the default value.

Preferably, the elongase and/or acyltransferase enzyme used in thepresent invention will not be native to the plant (i.e. the enzyme willbe foreign to the plant). Thus, the nucleic acid encoding the foreignenzymes may be referred to as a transgene. The plant hosting the nucleicacid encoding the foreign enzyme(s) will therefore be transgenic. Such aplant may be readily distinguished from native plants due to thepresence of foreign genetic material which would not be found in thenative, non-transgenic plant, for example, vector sequences, markergenes, and multiple copies of nucleic acid encoding one or both of theabove enzymes.

The transgene may encode an enzyme which is not present in the native,non-transformed plant, for example the 2-acyltransferase able toincorporate erucic acid at position 2 encoded by lat2 is not present innative, non-transformed Brassica napus. In such case a transgenic plantmay be distinguished from a native, non-transformed plant by an assayfor the foreign enzyme activity or by the presence of the reactionproduct (in this example trierucin). As the Limnanthes gene encodinglat2 is not present in native, non-transformed Brasica napus, atransgenic plant may also be distinguished from a native,non-transformed plant by testing for the presence of the lat2 genesequence e.g. by PCR or Southern blot.

Alternatively the transgene may encode an enzyme which is already foundin native, non-transformed plants, for example the elongase enzymesencoded by BnFAE1-1 and BnFAE1-2 are found in non-transformed Brassicanapus (because this is the plant from which the genes were isolated). Inthis case a transgenic plant may be identified by changed enzymeactivity (e.g. level, timing) in comparison with a non-transformedplant. Presence of the transgene may be positively verified by PCR orhybridisation tests for the presence of nucleotide sequences which areknown to be unique to the expression cassette containing the transgene.

Preferably, the nucleic acid sequences encoding the acyltransferaseand/or elongase are expressed in the plant through the period of oilbiosynthesis. This preferably includes at least the period during whichthe fatty acids are incorporated into the glycerol backbone, i.e.triacylglycerols are produced. It may also include the period duringwhich very long chain fatty acids are produced, the period of oilaccumulation in the plant, and/or the period of any oil modification.The timing of each of these stages of oil biosynthesis may be readilydetermined by a person skilled in the art using time-course experimentsusing well established biochemical methods, such as GLC (Gas LiquidChromatography) and HPLC ELSD (Evaporative Light Scatter Detection)analysis on triglycerides. The precise temporal expression pattern ofthe nucleic acid sequences may be adjusted according to their role inthe oil biosynthesis pathway. For example, it may be desirable toexpress the elongase enzyme prior to the start of oil biosynthesis,during the period of fatty acid synthesis, in order to effectivelyincrease the amount of “free” erucic acid prior to its incorporationinto lipid. Similarly, the acyltransferase enzyme may be best expressedduring the period of triacylglycerol synthesis. Depending upon thedegree of overlap between the different stages of oil biosynthesis, itmay be preferable for the elongase and acyltransferase enzymes to beexpressed at the same time. In a most preferred embodiment, the temporalexpression patterns of the foreign enzymes will mimic those of thecorresponding native enzymes of the plant.

In addition to conferring temporal specificity on the foreign enzymes,it may also be desirable to express them in a spatially specific manner,for example to reduce any adverse effects in parts of the plant notinvolved in oil biosynthesis. In a preferred embodiment, the elongaseand/or acyltransferase enzymes are expressed in the seed of the hostplant.

The desired temporal and spatial specificity may be conferred by the useof appropriate regulatory sequences to drive expression of the nucleicacid sequences encoding the elongase and acyltransferase enzymes. Use ofthese regulatory sequences will allow a tighter control of transgeneexpression and improved co-ordination with oil biosynthesis in seeds.This will enable potentiation of transgene activity whilst avoidingectopic expression. Thus, in a preferred embodiment, the elongase and/oracyltransferase nucleic acid sequences to be expressed in the plant areunder the control of one or more regulatory sequences capable of drivingexpression in a temporally and spatially specific manner. Depending uponthe desired temporal and spatial specificity of the acyltransferase andelongase enzymes, it may be preferably to place each under the controlof the same or separate regulatory sequences. Examples of suitableregulatory sequences include the oleosin promoter (Plant et al Plant MolBiol 25(2) 193-205 (1994)), the 2S napin promoter (European patent No0255278), and the FAE promoters of Brassica napus (Han et al., PlantMol. Biol. 46 229-239 (2001)) and Arabidopsis (Rossak et al., Plant Mol.Biol. 46 717-725 (2001)). The regulatory sequences which driveexpression of the native elongase and acyltransferase enzymes in theplant may also be used. The nucleic acid sequence to be expressed mayalso comprise 3′ polyadenylation sequences, for example the ChalconeSynthase polyadenylation termination signal sequence.

In achieving the desired levels of erucic acid in the oil of thetransgenic plant, it may be useful to overexpress the transgenes. Thus,the expression level will be higher than that of the native enzymeduring its active period. This may be achieved by placing the transgenesunder the control of a strong promoter, such as the napin promotes. TheFAE promoters of Brassica napus may also be suitable for overexpressionof the transgenes.

Where the elongase or acyltransferase enzymes are foreign to the plantinto which they are introduced, it may be desirable to down-regulate, ordisrupt the function of, the corresponding native enzymes or geneproducts in the plant. For this purpose, antisense sequences of thenative elongase or acyltransferase genes may be used to suppress theexpression of the native enzymes. The RNA transcribed from the antisenseDNA will be capable of binding to, and destroying the function of, asense version of the RNA found native in the cell, thereby disruptingits function. For example, antisense acyltransferase may be used in aplant where the native 2-acyltransferase is not specific for very longchain fatty acids such as erucic acid. Preferably, the antisensesequences will be capable of hybridising to the native sequence understringent conditions, as defined below.

It is not crucial for any such antisense sequence to be transcribed atthe same time as the nucleic acid encoding the foreign acyltransferaseand elongase. Antisense RNA will in general only bind when its sensecomplementary strand is present and so will only have its toxic effectat the appropriate time. Thus, any suitable plant promoter may be used,although it will preferably share the same spatial specificity as thenative enzyme. Examples of suitable promoters include the napin promoterand the promoter of the lat2 gene from limnanthes.

In the context of the present invention “stringent conditions” aredefined as those given in Plant genetic Transformation and GeneExpression: A Laboratory Manual, Ed. Draper et al 1988, BlackwellScientific Publications, p252-255, modified as follows:prehybridization, hybridization and washes at 55-65° C., final washes(with 0.5×SSC, 0.1% SDS) omitted.

Alternatively, ribozyme technology may be used to disrupt the expressionof the native elongase and/or acyltransferase in the plant. Ribozymesare RNA “enzymes” capable of highly specific cleavage against a giventarget sequence (Haseloff and Gerlarch, Nature 334 585-591 (1988)).

The nucleic acid sequences to be expressed in the plant may beintroduced in the form of a vector. Where nucleic acids encoding both anelongase and acyltransferase enzymes are to be introduced, it may bepreferable to incorporate both into a single vector. The vector may be,for example, a phage, plasmid or cosmid.

The vector will preferably also include one or more marker genes toenable the selection of plant cells which have been successfullytransformed. Examples of suitable marker genes include antibioticresistance genes such as those conferring resistance to kanamycin, G418and hygromycin (npt-II, hyg-B); herbicide resistance genes such as thoseconferring resistance to phosphinothricin and sulfonamide basedherbicides (bar and suI respectively; EP-A-242246 and EP-A-0369637) andscreenable markers such as beta-glucoronidase (GB2197653), luciferaseand green fluorescent protein.

The marker gene is preferably controlled by a second promoter whichallows expression in cells other than the seed, thus allowing selectionof cells or tissue containing the marker at any stage of development ofthe plant. Preferred second promoters are the promoter of nopalinesynthase gene of Agrobacterium and the promoter derived from the genewhich encodes the 35S subunit of cauliflower mosaic virus (CaMV) coatprotein. However, any other suitable second promoter may be used.

In order to assist the process of deregulation, it is preferred that theselectable marker gene is not present in the plant which is to be growncommercially. Various techniques for marker elimination are available,including co-transformation followed by segregation and selection ofsegregants having the gene of interest but lacking the marker gene.

Methods for introducing nucleic acid into a plant are known to personsskilled in the art, and include techniques such as by the use of adisarmed Ti-plasmid vector carried by agrobacterium, for example asdescribed in EP-A-0116718 and EP-A-0270822. Alternatively, the nucleicacid may be introduced by way of a particle gun, directly into the plantcells. This method is preferred for example where the plant is amonocot.

The oil may be extracted from the mature seeds of the plant by anysuitable means which will be known to persons skilled in the art. Forexample, the methods described in Wilmer et al J Plant Physiol 147486-492 (1996)) are available for laboratory scale extraction. Forlarger scale extraction, standard crushing techniques as practised bythe industry may be used.

In a second aspect of the present invention, there is provided a plantcapable of producing oil having an erucic acid content above 66%.Preferably, the plant is transgenic, and includes nucleic acid encodingan elongase enzyme capable of the production of very long chain fattyacids, including erucic acid, and nucleic acid encoding a2-acyltransferase. Most preferably, the transgenes are in accordancewith those defined in the first aspect of the invention.

The transgenic plants of the second aspect may be used in the productionof tailored oils, which differ from native oils of the plant. In thepresent invention, the oil of the transgenic plant will differ from thatof the native plant in terms of the erucic acid content of its oil. Inparticular, the erucic acid content of the oil will be higher than thatof the native plant, and most preferably, it will be above 66%.

In a preferred embodiment of the invention, one or more of the plants'native oil biosynthesis enzymes may be rendered inoperative.

Any plant may be used in the present invention. Preferred plants arethose whose seeds are used in the production of oil, for exampleBrassica napus, other Brassica, mustards, or other cruciferous plants,sunflower, soya or maize.

Also provided are plant parts, such as material required forpropagating, in particular seeds.

A whole plant can be regenerated from a single transformed plant cell.Thus, in a further aspect the present invention provides a transgenicplant cell including nucleic acid sequences encoding an elongase enzymecapable of the production of very long chain fatty acids, includingerucic acid, and nucleic acid encoding a 2-acyltransferase. Theregeneration can proceed by known methods (for Brassica napus seeMoloney et al Plant Cell Reports 8 238-242, 1989).

In a third aspect of the invention, there is provided oil having anerucic acid content above 66%. Preferably, the oil is produced by amethod of the first aspect. In a final aspect of the present invention,there is provided the use of such a transformed plant in the productionof tailored oil.

Preferred features of the first aspect of the invention are for theother aspects mutatis mutandis.

EXAMPLES Example 1 Isolation of BnFAE1-1 and BnFAE1-2 and Cloning into aBinary Vector

Starting Material

High erucic rape plants of a line BGRV2 were grown in the glasshouse toprovide leaf material for DNA isolation. DNA was isolated essentially asdescribed in Sambrook et al. (Molecular cloning: a laboratory manual,2^(nd) ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor,N.Y., 1989).

PCR Amplification of BnFAE1 Sequences

Previously published B.napus fae1 sequences were analysed and twooligonucleotide primers were designed to amplify the B.napus fae1 genesfrom the high erucic acid winter line BGRV2: (SEQ ID NO:4) BnFAE-F5′CCTCATGACGTCCATTAACGTAAAGCTCC 3′ and (SEQ ID NO:5) BnFAE-R5′GTGAGCTCTTATTAGGACCGACCGTTTGGG 3′.

PCR was performed using Tli Taq polymerase in the buffer supplied with1.5 mM MgCl2, and an annealing temperature of 60° C. PCR products werecloned into pBluescriptII KS(+) (Stratagene) SmaI site and sequenced(MWG Biotech). (FIGS. 1 and 2)

The sequence of the BnFAE1-1 open reading frame shows higher homology toa published partial cDNA derived from Brassica oleracea than doesBnFAE1-2. We therefore suggest that BnFAE1-1 represents the gene derivedfrom the B. oleracea parent of B. napus (C-genome) and that BnFAE1-2represents the B. rapa genome (A-genome).

Construction of Plasmids pEW13 and pEW14

The two fae1 genes which had been amplified, cloned into pBluescript II,and sequenced were designated as pEW1 (fae1-1) and pEW3 (fae1-2). Thefae1 genes were transferred as RcaI-EcoICRI fragments into pAR4(Biogemma UK) NcoI-SmaI sites to place the genes between the Pnapin andCHS polyA sequences (pEW7 and 6). The two fae1 expression cassettes wereinserted adjacent to a similar Lat2 cassette, by digesting pEW7 and 6with SalI-SacI and inserting the fragments into pT7Blue2 SalI-SacI sites(creating pEW9 and 8), before transfer of an EcoRI EcoICRI fragment intothe binary vector pSCVnos144 (Biogemma UK) SalI-SmaI sites resulting inpEW13 and 14. The cloning strategy for the construction of pEW13 isshown in FIG. 4.

Example 2 BnFAE1 Genes Encode Elongase Activities which SpecificallyCatalyse the Formation Erucic Acid (22:1)

To demonstrate the product specificity of the two BnFAE1 genes, theywere expressed in developing embryos of low erucic acid rape (LEAR).

Both plasmid pEW13 and plasmid pEW14 were transformed into Agrobacteriumstrain C58pMP90 and transformed into LEAR using agrobacterialtransformation essentially as described in Moloney et al., (Plant CellReports 8: 238-242, 1989). Seed of the resulting transformed plantscontain significant levels of erucic acid since both BnFAE1 genescomplement mutations in the elongases of the LEAR line.

Oil extraction, separation, and analysis by GC was performed essentiallyas described in Wilmer et al (J. Plant Physiol. 147: 486-492, 1996). Oilwas extracted from samples of 20 seed from each self-pollinatedtransformed plant. Seed were extacted with 1 ml chloroform, 2.8 mlmethanol/0.01 N hydrochloric acid (1:0.8, by vol.), containing 2 mg/mltriheptadecanoin as an internal standard. After shaking, 1 ml chloroformand 1 ml 0.01N hydrochloric acid were added and mixed. The phases wereseparated by centrifugation in a bench-top centrifuge, and the aqueousphase washed with a further 1.5 ml chloroform. The organic fractioncontains most lipids.

Triglycerides were purified on 0.25 mm Silica gel 60 TLC plates,developed in hexane:diethyl ether:acetic acid (70:30:1 by vol.). Afterdrying the lipids were visualised with iodine vapour and areascontaining triglycerides were scraped off. TAG were transmethylated toproduce fatty acid methyl esters (FAMEs) which could be analysed by gaschromatography.

FAME oil analysis was performed on mature seed of 11 plants transformedwith constructs containing the fae1-1 gene and 25 plants transformedwith constructs containing the fae1-2 gene demonstrating that theintroduction of either fae1 gene led to a dramatic increase in theVLCFAs eicosenoic and erucic acid (C20:1 and C22:1) with a correspondingdecrease in oleic acid (C18:1) (FIG. 5). Both fae1 genes functioned wellin the production of VLCFAs.

Example 3 BnFAE1 Genes Encode Elongase Activities which Increase ErucicAcid Levels in High-Erucic Acid Oilseed Rape

A winter high-erucic acid line, BGRV2 was transformed with the twoplasmids pEW13 and pEW14 previously tested in LEAR to examine whetherthe yield of erucic acid could be further enhanced in a cultivar whichproduced erucic acid.

Both plasmid pEW13 and plasmid pEW14 were transformed into Agrobacteriumstrain C58pMP90 and transformed into HEAR using agrobacterialtransformation essentially as described in Moloney et al., (Plant CellReports 8: 238-242, 1989).

Oil analysis was performed on mature seed as described in Example 2.

Twenty seven plants were analysed (10 with fae1-1 plus lat2, 17 withfae1-2 plus lat2), 9 of which showed an increase in erucic acid of atleast 4 mol %, the highest with 67.5 mol % fatty acids compared to thecontrol of 54% as shown in FIG. 6. Southern blot hybridisation of someof these plant lines showed that those with an increase in erucic acidhad single or double insertions, whereas one of three lines whichexhibited a considerable decrease (34%) contained 5 copies (data notshown), so that the phenotype observed could be due to co-suppression.

The data were applied to a t-test to determine whether there was asignificant difference between the two populations of plants derivedfrom fae1-1 or fae1-2 plus lat2. The probability value p=0.00056917,demonstrated that there was a significant difference between thepopulations, and that fae1-2 appeared to be more efficient.

The construct pEW14 (fae1-2 plus lat2) was also used to transform aCanadian spring HEAR line BGRV40, with similar results. Three of thefour transgenic plants produced, exhibited a considerable increase inerucic acid whereas one line was repressed.

In the lines derived from either of the HEAR varieties it is C22:1 whichis specifically increased, not C20:1.

In all cases, it is the Ti generation that has been analysed, which ishemizygous. After self-fertilization and segregation analysis it will bepossible to re-evaluate the erucic acid levels in the T2 generation,which may then exhibit further increases in C22:1 as the inserted T-DNAbearing the elongase and acyl-transferase cassette becomes homozygous.

Sn-2 analysis confirmed the incorporation of C22:1 at the Sn-2 positionof the TAG by the 2-acyltransferase encoded by the introduced lat2 gene.

Example 4 Very High Erucic Acid Levels are Stable in the next Generationin Oilseed Rape

Five lines of the winter high-erucic acid line, BGRV2, transformed withthe plasmid pEW 14, and three lines of the spring high-erucic acid line,BGRV40, transformed with the plasmid pEW14, chosen from the materialdescribed in Example 3 were grown in a following generation.

Four of the winter high erucic-acid lines and two of the springhigh-erucic acid lines contained one or two copies of the transgene asdetermined by southern analysis, whereas one each of the winter andspring lines contained multiple copies of the transgenes.

Twenty plants per line were grown in the glasshouse and presence of theadditional fael-2 gene was confirmed using PCR with a primer sequenceinternal to the fael-2 sequence and a primer inside the CHS terminatorsequence. Mature seed was collected for analysis and oil analysis wasperformed on these seed as described in Example 2.

The two lines with multiple copies of the transgene that showed reducedlevels of erucic acid in the T1 seed, both produced a wide range oferucic levels in the T2, as shown in FIG. 7.a and b. The lowest levelsobserved were below those observed in T1 seed, the highest levels wellabove the level of erucic acid observed in non-transgenic controloilseed rape of the appropriate line, and similar to the highest levelsobserved in the low-copy number lines, at least for the winter oilseedrape.

For the lines that showed increases in erucic acid in the T1 seed, thisphenotype was maintained in the T2 seed, with the highest levels oferucic acid observed still around 67 to 68% (FIG. 7.c and d).

When the progeny of the lines with single or double copies of thetransgene were tested by PCR for the presence of the transgene,segregation ratios of transgenic versus non-transgenic plants wereobserved that are consistent with a mendelian segregation of thetransgene. Chi-square tests returned values showing non-significantdeviation from an independent segregation model (data not shown).

1. A method of producing, in a plant, oil having an erucic acid contentabove 66% erucic acid, the method comprising (i) expressing in the plantnucleic acid encoding an elongase and nucleic acid encoding anacyltransferase enzyme; and (ii) extracting oil from the plant.
 2. Amethod according to claim 1, wherein the elongase is a fatty acidelongase enzyme from Brassica napus, or one substantially identicalthereto.
 3. A method according to claim 2, wherein the fatty acidelongase is encoded by the sequence of FIG. 1 or 2, or a sequencesubstantially identical thereto.
 4. A method according to claim 1,wherein the acyltransferase is a 2-acyltransferase.
 5. A methodaccording to claim 4, wherein the 2-acyltransferase is from L.douglassi.
 6. A method according to claim 1, wherein the nucleic acidsequences are under the control of a regulatory sequence.
 7. A methodaccording to claim 6, wherein the regulatory sequence is an FAE promoterof Brassica napus.
 8. A method according to claim 1, wherein the nucleicacid sequences are provided in the form of a vector.
 9. A methodaccording to claim 1, wherein the nucleic acid sequences are operablylinked to one or more marker genes.
 10. A method according to claim 1,wherein the activity of the native elongase and/or acyltransferase ofthe plant is suppressed.
 11. A method according to claim 10, wherein theactivity is suppressed by antisense technology.
 12. A transgenic plantcapable of producing oil having an erucic acid content above 66%.
 13. Atransgenic plant according to claim 12 which expresses a2-acyltransferase and an elongase enzyme which are not native to theplant.
 14. A plant according to claim 12 which is Brassica napus.
 15. Aseed of a plant of claim
 12. 16. Oil produced by a method of any one ofclaims
 1. 17. Oil extracted from a plant of claim 12.